Project 11: Retinoblastoma Protein Inactivation Results in a Metabolic Shift Toward Glutamine Dependency - Brian F. Clem, Ph.D.

Retinoblastoma protein (Rb), the first described tumor suppressor, plays a vital role in regulating cell proliferation by modulating the expression of key genes involved in cell cycle progression.  Although the majority of work investigating the role of Rb function in tumorigenesis has focused on cell cycle regulation, it is clear that Rb may mediate a broad spectrum of biochemical pathways, particularly those involved in cellular metabolism.  Dysregulated metabolism is now considered a hallmark of tumorigenic cells, as evident by increased glucose utilization as well as a dependence on glutamine consumption.  Specifically, glutamine serves to fulfill the metabolic need for energy production, nucleotide and amino acid synthesis, and the production of glutathione.  Even though specific enzymes have been implicated in this phenomenon of “glutamine addiction” in tumor cells, the precise signaling mechanisms and the oncogenic factors that facilitate this metabolic shift have yet to be fully described.  Using genetically depleted Rb knockout mouse embryonic fibroblasts, we demonstrate that Rb Inactivation specifically leads to dependence on glutamine for cell growth and survival.  Our data further suggest that this metabolic shift may be through a c‐Myc dependent pathway, as Rb depletion increases both c‐Myc transcript and protein levels.  At the completion of this proposal, we expect to have fully characterized the regulation of glutamine metabolism by Rb and, in doing so, provide a greater understanding of the nature of the neoplastic phenotype, especially with respect to disruption of the Rb network.

We will test our hypothesis by conducting the following Specific Aims:

Specific Aim 1.  Determine the specific pathways involved in increased glutamine utilization between wild-type and Rb-depleted mouse embryonic fibroblasts. Rb-mediated glutamine metabolism will be examined in either wild-type mouse embryonic fibroblasts (WT-MEF) or MEF cells lacking the three members of the Rb family (TKO-MEF).  Baseline glutamine utilization within these distinct cell types will be determined using 13C-glutamine stable isotopomer analysis by NMR.  Additionally, the effects of potential regulators of glutamine utilization, such as c-Myc and glutaminase, will be investigated by specific shRNA knockdown.

Specific Aim 2.  Determine the effects of glutaminase function on the survival, growth, and oncogenic transformation of Rb-depleted mouse embryonic fibroblasts. The survival and growth sensitivities of WT and Rb-deficient MEFs to glutaminase expression and activity will be assessed by lentivirus-mediated shRNA stable knockdown in both cell types.  In each scenario, cells will be analyzed for:  (i) cell viability; (ii) proliferation; and (iii) anchorage-independent growth in soft agar.  In addition, the effects of shRNA specific knockdown of glutaminase in xenografts of Rb-deficient TKO MEF’s will also be utilized to assess glutaminase inhibition on Rb-mediated tumorigenic growth in vivo.  Established tumors will be examined for in situ growth using micro-CT imaging, and for viability and apoptosis by H&E, Ki-67 nuclear proliferation antigen and cleaved caspase 3 staining.